Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.

Department of Cellular and Molecular Neuroendocrinology Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

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CLYNE JD, WANG LF, HUME RI. Mutational analysis of the conserved cysteines of the rat P2X2 purinoceptor. J Neurosci. 2002;22:3873–3880. PubMed PMC

CODDOU C, ACUNA-CASTILLO C, BULL P, HUIDOBRO-TORO JP. Dissecting the facilitator and inhibitor allosteric metal sites of the P2X4 receptor channel: critical roles of CYS132 for zinc potentiation and ASP138 for copper inhibition. J Biol Chem. 2007;282:36879–36886. PubMed

ENNION S, HAGAN S, EVANS RJ. The role of positively charged amino acids in ATP recognition by human P2X1 receptors. J Biol Chem. 2000;275:29361–29367. PubMed

ENNION SJ, EVANS RJ. Conserved cysteine residues in the extracellular loop of the human P2X1 receptor form disulfide bonds and are involved in receptor trafficking to the cell surface. Mol Pharmacol. 2002;61:303–311. PubMed

FABBRETTI E, SOKOLOVA E, MASTEN L, D'ARCO M, FABBRO A, NISTRI A, GINIATULLIN R. Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+ sensing modulatory sites. J Biol Chem. 2004;279:53109–53115. PubMed

FRIDAY SC, HUME RI. Contribution of extracellular negatively charged residues to ATP action and zinc modulation of rat P2X2 receptors. J Neurochem. 2008;105:1264–1275. PubMed PMC

JARVIS MF, KHAKH BS. ATP-gated P2X cation-channels. Neuropharmacology. 2009;56:208–215. PubMed

JELINKOVA I, YAN Z, LIANG Z, MOONAT S, TEISINGER J, STOJILKOVIC SS, ZEMKOVA H. Identification of P2X4 receptor-specific residues contributing to the ivermectin effects on channel deactivation. Biochem Biophys Res Commun. 2006;349:619–625. PubMed

JIANG LH, RASSENDREN F, SURPRENANT A, NORTH RA. Identification of amino acid residues contributing to the ATP-binding site of a purinergic P2X receptor. J Biol Chem. 2000;275:34190–34196. PubMed

KAWATE T, MICHEL JC, BIRDSONG WT, GOUAUX E. Crystal structure of the ATP-gated P2X4 ion channel in the closed state. Nature. 2009;460:592–598. PubMed PMC

KHAKH BS, NORTH RA. P2X receptors as cell-surface ATP sensors in health and disease. Nature. 2006;442:527–532. PubMed

KHAKH BS, PROCTOR WR, DUNWIDDIE TV, LABARCA C, LESTER HA. Allosteric control of gating and kinetics at P2X4 receptor channels. J Neurosci. 1999;19:7289–7299. PubMed PMC

NICKE A, BAUMERT HG, RETTINGER J, EICHELE A, LAMBRECHT G, MUTSCHLER E, SCHMALZING G. P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. Embo J. 1998;17:3016–3028. PubMed PMC

NORTH RA. Molecular physiology of P2X receptors. Physiol Rev. 2002;82:1013–1067. PubMed

PRIEL A, SILBERBERG SD. Mechanism of ivermectin facilitation of human P2X4 receptor channels. J Gen Physiol. 2004;123:281–293. PubMed PMC

RASSENDREN F, BUELL G, NEWBOLT A, NORTH RA, SURPRENANT A. Identification of amino acid residues contributing to the pore of a P2X receptor. Embo J. 1997;16:3446–3454. PubMed PMC

ROBERTS JA, EVANS RJ. ATP binding at human P2X1 receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists. J Biol Chem. 2004;279:9043–9055. PubMed

ROBERTS JA, EVANS RJ. Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP. J Neurochem. 2006;96:843–852. PubMed

SILBERBERG SD, LI M, SWARTZ KJ. Ivermectin Interaction with Transmembrane Helices Reveals Widespread Rearrangements during Opening of P2X Receptor Channels. Neuron. 2007;54 :263–274. PubMed

SURPRENANT A, NORTH RA. Signaling at purinergic P2X receptors. Annu Rev Physiol. 2009;71:333–359. PubMed

TOULME E, SOTO F, GARRET M, BOUE-GRABOT E. Functional properties of internalization-deficient P2X4 receptors reveal a novel mechanism of ligand-gated channel facilitation by ivermectin. Mol Pharmacol. 2006;69:576–587. PubMed

YAN Z, LIANG Z, OBSIL T, STOJILKOVIC SS. Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate. J Biol Chem. 2006;281:32649–32659. PubMed

YAN Z, LIANG Z, TOMIC M, OBSIL T, STOJILKOVIC SS. Molecular Determinants of the Agonist Binding Domain of a P2X Receptor Channel. Mol Pharmacol. 2005;67:1078–1088. PubMed

YI CL, LIU YW, XIONG KM, STEWART RR, PEOPLES RW, TIAN X, ZHOU L, AI YX, LI ZW, WANG QW, LI CY. Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X4 receptors. Biochem Biophys Res Commun. 2009;381:102–106. PubMed

ZEMKOVA H, YAN Z, LIANG Z, JELINKOVA I, TOMIC M, STOJILKOVIC SS. Role of Aromatic and Charged Ectodomain Residues in the P2X4 Receptor Functions. J Neurochem. 2007;102:1139–1150. PubMed

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking